NCCa-ATP Channel
A unique non-selective monovalent cationic ATP senstive channel (NCCa-ATP channel) was identified first in native reactive astrocytes (NRAs) and later, as described herein, in neurons and capillary endothelial cells after stroke or traumatic brain injury (See, International application WO 03/079987 to Simard et al., and Chen and Simard, 2001, each incorporated by reference herein in its entirety). The NCCa-ATP channel is thought to be a heteromultimer structure comprised of sulfonylurea receptor type 1 (SUR1) regulatory subunits and pore-forming subunits, similar to the KATP channel in pancreatic β cells (Chen et al., 2003). The pore-forming subunits of the NCCa-ATP channel remain uncharacterized.
SUR imparts sensitivity to antidiabetic sulfonylureas such as glibenclamide and tolbutamide, and is responsible for activation by a chemically diverse group of agents termed “K+ channel openers” such as diazoxide, pinacidil and cromakalin (Aguilar-Bryan et al., 1995; Inagaki et al., 1996; Isomoto et al., 1996; Nichols et al., 1996; Shyng et al., 1997). In various tissues, molecularly distinct SURs are coupled to distinct pore-forming subunits to form different KATP channels with distinguishable physiological and pharmacological characteristics. The KATP channel in pancreatic β cells is formed from SUR1 linked with Kir6.2, whereas the cardiac and smooth muscle KATP channels are formed from SUR2A and SUR2B linked with Kir6.2 and Kir6.1, respectively (Fujita et al., 2000). Despite being made up of distinctly different pore-forming subunits, the NCCa-ATP channel is also sensitive to sulfonylurea compounds.
Also, unlike the KATP channel, the NCCa-ATP channel conducts sodium ions, potassium ions, cesium ions and other monovalent cations with near equal facility (Chen and Simard, 2001) suggesting further that the characterization, and consequently the affinity to certain compounds, of the NCCa-ATP channel differs from the KATP channel.
Other nonselective cation channels that are activated by intracellular Ca2+ and inhibited by intracellular ATP have been identified but not in astrocytes. Further, the NCCa-ATP channel expressed and found in astrocytes differs physiologically from the other channels with respect to calcium sensitivity and adenine nucleotide sensitivity (Chen et al., 2001).
Other nonselective cation channels that are activated by intracellular Ca2+ and inhibited by intracellular ATP have been identified in endothelial cells (Csanady and Adam-Vizi, Biophysical Journal, 85:313-327, 2003), but these channels are not regulated by SUR1 and are not inhibited by glibenclamide.
Spinal Cord Injury
A contusion injury to the spinal cord is often worsened by secondary damage from tissue inflammation and swelling. Secondary injury that expands the region of irreversible damage should, in principal, be preventable since it occurs in delayed fashion while under medical care, but effective treatments are not yet available. Secondary injury typically involves a zone of potentially viable tissue, called the penumbra, that surrounds the initial injury. Viability of neural tissues in the penumbra is precarious, and those tissues can easily succumb and die.
Changes in gene expression related to inflammation are among the earliest and strongest responses following spinal cord injury (Bareyre and Schwab, 2003; Bartholdi and Schwab, 1997).
An inflammatory response is necessary for resolution of the pathogenic event, but bystander or collateral tissue damage is caused by the toxic nature of many of its by-products. It is generally recognized that inflammation can be deleterious because cytotoxic agents such as TNFα and NO may be released, and because inflammation promotes formation of edema and swelling, which in turn contribute to tissue ischemia. Thus, a strong inflammatory response can cause expansion of the original zone of tissue death. In contrast, ameliorating the inflammatory response can diminish the overall extent of damage.
One of the most potent stimulators of inflammation in spinal cord injury is blood that extravasates from fractured capillaries following injury. Blood is universally held to be highly toxic to central nervous system tissues, include spinal cord.
Cells die by apoptosis and necrosis. The distinction is important, not so much for cells that die, but for cells in surrounding tissues—the penumbra—that may survive, albeit tenuously at first. Necrotic death incites an inflammatory response, whereas apoptotic death does not. Molecular mechanisms responsible for inflammation following necrotic cell death are not fully understood, but it is likely that necrotic death, unlike apoptotic death, is accompanied by release of intracellular molecules when cell membranes lyse. These intracellular molecules, when released, activate other cells, notably microglia, whose activation results in expression of chemokines that in turn attract inflammatory cells. Thus, a logical therapeutic goal is to reduce necrosis, even if only to convert it to apoptosis, to reduce the release of intracellular molecules that initiate inflammation.
An important class of intracellular molecules that can initiate inflammation in necrotic death is heat shock proteins (HSP). Injury to the spinal cord causes activation of astrocytes and up-regulation of developmentally regulated intracellular proteins, including vimentin, nestin and HSP. HSP-32 and HSP-70 are of special interest because they are up-regulated in spinal cord injury (Song et al., 2001; Mautes et al., 2000; Mautes and Noble, 2000). In astrocytes, HSP-32 (heme oxygenase-1) is induced by blood and blood products, and HSP-70 is induced by hypoxia or glucose deprivation (Regan et al., 2000; Matz et al., 1996; Lee et al., 2001; Currie et al., 2000; Xu and Giffard, 1997; Papadopoulos et al., 1996; Copin et al., 1995).
HSP-70 and HSP-32 activate microglia in vivo, (Kakimura et al., 2002) and activated microglia, in turn, release inflammatory chemokines that attract macrophages and polymorphonuclear leukocytes (PMNs). Thus, deleterious pathological events leading to inflammation-mediated secondary injury may originate, in part, with necrotic death of astrocytes and release of HSPs as well as from extravasated blood. Therefore, the present invention is directed to decreasing necrotic death of reactive astrocytes and to reducing extravasation of blood as an improved therapeutic strategy to treat spinal cord injury.